It's been soooo looong....

...but I'm about to do a real experiment at the bench, because one of my post-docs has called me on my bragging that "I can do a time course in my sleep".

Several years ago we did some collaborative work on DNA uptake by Actinobacillus pleuropneumoniae, a H. influenzae relative that infects pigs rather than people (the PDF is here). The collaborators also generated data on the regulation of competence by the A. pleuropneumoniae homolog of our favourite protein, Sxy. They have now moved on to other things, and this post-doc is writing up a short manuscript (just a 'Note') combining their data with some of ours.

One issue her manuscript examines is how competence changes in response to different culture conditions. I had investigated this during the period of collaboration, by following changes in transformation frequency over time, as cells were grown in rich medium and transfered to the starvation medium that induces competence in H. influenzae. I did versions of this 'time course' several times, but each was plagued by inconsistencies in the numbers of colonies that grew up on the agar plates. I couldn't figure out what was causing the inconsistencies -- the cells were no more sensitive to minor variations in plating conditions than H. influenzae is -- so I compensated by sampling the culture more often. This generated data that was marginally acceptable, and I went on to other things.

Now the post-doc wants to include this data in her manuscript, but it needs to be replicated first. Over to me. I checked yesterday and was reassured to find that I have lots of the kanamycin-resistance DNA I need to give the cells (I sensibly did a large high-quality prep in 2005), and a good stock of frozen competent A. pleuropneumoniae cells that I can use as controls. So today I'm going to thaw out some of these cells and transform them with the DNA, just to confirm that everything is working. Sunday I'll count the colonies from that little experiment, and if all looks good I'll plan the big time course, using my best 2005 time course as a guide.

Monday I'll have help from our work-study student. She and I will make the agar for all the plates we'll need, and pour about 250 plates. She'll first make up a high-concentration stock of the NAD supplement this agar needs. We'll prepare lots of dilution solution and put 5 ml aliquots into hundreds of tubes. We'll start an overnight culture of the cells, so they'll be ready to go on Tuesday morning.

Tuesday I'll start by diluting the cells into fresh medium and following their growth by measuring the turbidity of the culture. If I'm lucky the work-study student will be free to help me. We'll put a drop (10 ul = 1 ug) of DNA in each of about 30 tubes. At about 30-minute intervals we'll take 1 ml of cells from the culture into a tube, incubate this for 15 min, add 10 ul DNaseI to destroy the remaining DNA, incubate for 5 minutes, and then dilute the cells and spread them on plates with and without kanamycin. Once the culture has reached a specific density we'll transfer part of it to starvation medium, and also test aliquots from this at 30 min intervals.

If we're ambitious we may also add the potential inducer cyclic AMP (cAMP) to another part of the culture. In H. influenzae cAMP induces competence, but my 2005 results with A. pleuropneumoniae were very unreproducible.

Then, on Wednesday or Thursday, maybe I can convince the work-study student that counting colonies is a thrilling part of scientific research (like Tom Sawyer white-washing the fence). Otherwise I'll have to count them myself.